Project Summary/Abstract Two-component systems are major signaling pathways bacteria use to sense diverse stimuli such as temperature, osmotic changes, and antibiotics and to initiate adaptive responses. In these systems the histidine kinase (HK) detects the stimulus and relays the signal to its cognate response regulator, which alters gene expression. As a postdoctoral scholar in Dr. William DeGrado's laboratory at the University of California, San Francisco (UCSF), I have been designing protein constructs to study the structural and conformational dynamics of HKs. Here, I propose to use an integrative experimental approach combining bacterial reporters, enzymatic assays, x-ray structural studies, and droplet-based microfluidics technologies to understand how conformational transitions in histidine kinases facilitate signal transduction. Recent HK structures suggest that symmetry across the dimer interface is intricately related to catalytic state. The goal of this proposal is to develop a molecular description of how structural signals induce symmetric to asymmetric conformational transitions in the catalytic, cytoplasmic regions of HKs. I hypothesize that bistability of the dimer interface `backbone' confers an essential conformational flexibility, which allows localized helical buckling to occur. The consequence of this design is a transition from a continuous helical path along the backbone in the symmetric state to a discontinuous path, which produces asymmetry. In Aim 1 I will examine how residue changes in the buckling region of the backbone affects HK signaling. In Aim 2 I will use protein design to determine the structural states associated with signaling in the cytoplasmic region of HKs. In Aim 3 I examine how signals transmitted into the cytoplasmic region of HKs become modulated by coupling between effector domains and the catalytic core. Completion of these aims will provide a molecular description of the structural and conformational dynamics of HKs. A better understanding of the structural states and conformational changes associated with signaling can inform structure-based design of small molecule inhibitors. By performing the research in this proposal, I will increase my proficiency in protein design and biophysics while simultaneously receiving strong training in X-ray crystallography, microfluidics, and high-throughput approaches to biology. Expertise in these areas will better allow me to pursue my long-term scientific goals of using protein design as a method to elucidate protein structure and function. The experience I will receive by working with my mentor Dr. DeGrado and my collaborators during the K99 phase of the award will help me to become a stronger scientist and better prepare me for a career as an independent researcher. The data and publications that result from doing the work in this proposal will make me a stronger faculty candidate and help with future grant applications. In general, completion of the research and training proposed in my application will provide me with the skill set necessary for achieving my long-term career goal of becoming a principal investigator.